Human antihuman interleukin-6 antibody and fragment of antibody

ABSTRACT

A substance effective for treating immunopathy where interleukin 6 (IL-6) is involved is provided. A human anti-human IL-6 antibody and a human anti-human IL-6 antibody fragment having a high affinity to human IL-6 were obtained using phage antibody technique. Said antibody and antibody fragment are expected to be useful as a medicament for treating inflammation and immunopathy caused by IL-6.

TECHNICAL FIELD

The present invention relates to a human anti-human interleukin-6(hereinafter referred to as “IL-6”) antibody that binds to human IL-6 tothereby block binding between IL-6 and its receptor, a fragment of saidantibody, and a gene fragment encoding the same. The antibody and afragment thereof according to the present invention are expected to beuseful as a medicament for treating inflammation and immunopathy causedby IL-6.

BACKGROUND ART

IL-6 is a glycoprotein with a molecular weight of 21,000 that isproduced from T cells, macrophages, fibroblasts, muscular cells and thelike when stimulated with a mitogen, viral infection, or IL-1. HumanIL-6 consists of 184 amino acids and its gene is present on the 7thchromosome. IL-6 has diverse biological activities including (1)induction of cellular proliferation (hybridomas, T cells, keratinocytes,renal mesangial cells), (2) inhibition of cellular proliferation(myelogenic leukemia cell lines, malignant melanoma cell lines), and (3)induction of cellular differentiation and induction of production ofcellular specific proteins (neural differentiation of melanocytoma celllines, differentiation of killer T cells, maturation of megakaryocytes,differentiation into macrophages of myelogenic leukemia cell lines,antibody production of B cells, production of acute phase proteins inhepatocytes). Due to its diverse biological activities, it has beenindicated that IL-6 may be relevant to some diseases. In recent years,it is known that IL-6 is involved in onset of diseases including (1)rheumatoid arthritis, atrial myxoma, Castleman disease,hypergammaglobulinemia or autoimmune symptoms in AIDS, (2) mesangialnephritis, (3) psoriasis, and (4) Kaposi sarcoma in AIDS. Recently, itis also known that a large quantity of IL-6 is produced from theskeletal muscle immediately after physical practice, which stimulateshypothalamus to secrete various neurohormones to thereby affect theimmune system (Dictionary of Immunology, 1st ed., p. 49, 1993).

Among the diseases where IL-6 is involved, rheumatoid arthritis (RA)afflicts about 7×10⁵ people all over the country in Japan with gradualincrease and together with increase in the number of aged patients isbecoming a social problem (Ogata A. et al., Rinsho Byori (ClinicalPathology), 1999 April; 47 (4): 321-326 [Advances in interleukin-6therapy]).

The cause of RA is not known. RA, an autoimmune disease wherein anautoimmune reaction within the articular cavity has continued and becamechronic, is assigned as one of inveterate specific diseases. Relevancyof RA to IL-6 has been investigated to reveal that a large quantity ofIL-6 is present in joint fluid from RA patients and that IL-6 isinvolved not only in induction of inflammation but also in proliferationof fibroblasts in the synovial membrane. There is also possibility thatIL-6 may accelerate production of autoantibody (Nishimoto N. et al.,Clinical application of interleukin-6 receptor antibody, transactions ofJapanese Society for Immunology 1997; 20: 87-94).

Accordingly, anti-IL-6 antibody that inhibits the biological activitiesof IL-6 would be a candidate of a nosotropic medicament for treatingseveral immunopathies including RA and is practically underinvestigation (Mihara M. et al., Br. J. Rheumatol. 1995 April; 34(4):321-325; Mihara M. et al., Clin. Immunol. 2001, 98: 319-326).

DISCLOSURE OF THE INVENTION

(Technical Problems to be Solved by the Invention)

For RA patients, a wide variety of treatments have been appliedincluding drug therapy with non-steroidal antiinflammatory, analgesicagents, steroidal agents, immunosuppressive agents or antimetabolites,and surgical therapy such as artificial joint, depending on a diseasestage of patients. However, these therapies are not eradicative for RAbut there are problems of adverse side effects due to application oftherapies for a long period of time with a large amount of drugs. IL-6plays a role in enhancement of inflammation and hence is a major causeof pain RA patients suffered from. It has been indicated therefore thatinhibition of the IL-6 activity would alleviate the pain. As acandidate, a humanized anti-IL-6 antibody has been investigated(Montero-Julian F. A. et al., Blood 1995 Feb. 15; 85(4): 917-24; MonierS. et al, Clin. Exp. Rheumatol. 1994 November-December; 12(6): 595-602;Wendling D. et al, J. Rheumatol. 1993 February; 20(2): 259-62).

On the other hand, IL-6 has an activity of a growth factor to myelomacells (Dictionary of Immunology, 1st ed., p. 49, 1993; aforementioned)and hence causes a problem that, even if hybridomas producing anantibody that binds to IL-6 with high affinity were obtained, theirproliferation is hampered through neutralization of IL-6 in the culturemedium by the produced antibody and as a result obtaining an anti-IL-6antibody with high affinity has been difficult. Sato et al. reportedthat an anti-human IL-6 antibody obtained from mice exhibited highaffinity of 11 nM but also with a high dissociation rate of 3×10⁻² sec.(Sato K. et al., Hum. Antibodies Hybridomas 1996; 7(4): 175-83). Withsuch an antibody having a high dissociation rate as obtained by theprior art techniques, maintenance of a high concentration of theantibody was necessary for inhibiting the IL-6 activity. Much less, anantibody with such an activity is never known that is a wholly humanantibody.

Besides, unlike a wholly human antibody, a possibility could not bedenied that administration of a humanized antibody to patients wouldlead to production in patients of an antibody (blocking antibody) thatinhibits the activity of the anti-IL-6 antibody.

(Means to Solve the Problems)

Under the circumstances, the present inventors devised a screeningsystem with the phage antibody technique to thereby obtain a whollyhuman anti-human IL-6 antibody single chain Fv (scFv) molecule andelucidated VH and VL chains of said antibody. The present inventorsfurther analyzed the properties of said scFv to reveal that said scFvexhibited a significantly lower association rate as compared to those ofthe conventional antibodies against human IL-6 obtained from a varietyof animals (in the order of 10⁻³ sec; dissociation rate being about40-folds lower than that of conventional ones), had an equivalent orhigher affinity to IL-6 as compared to the conventional antibodies, andinhibited proliferation of IL-6 dependent cell lines in a concentrationdependent manner.

(More Efficacious Effects than Prior Art)

It is expected that the use of such an antibody that is wholly derivedfrom human and has a high affinity to IL-6 would exert therapeuticeffects with a lower antibody concentration than a chimeric antibody ora humanized antibody to thereby produce only an extremely low level ofanti-idiotype antibody against said antibody and hence would provide ananti-human IL-6 antibody drug that will exhibit excellent therapeuticeffects as an anti-IL-6 antagonist for treating autoimmune diseases suchas IL-6 dependent leukemia and rheumatoid arthritis. The antibodyaccording to the present invention is also expected for use as amedicament for treating acute inflammation with reduced side effects andwith potent activity.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the results of ELISA where reactivity ofIL6gk3-2scFv from IL-6gk series with a recombinant IL-6, human serumalbumin (HSA), AB blood type serum, monocyte chemoattractant protein-1(MCP-1) and MIP-1α (macrophage inflammatory protein-1α) was measured.

FIG. 2 is a graph showing the results of BIA CORE where a bindingaffinity of IL6gk3-2 scFv with IL-6 was measured.

FIG. 3 is a graph showing the results that IL6gk3-2 scFv inhibited IL-6dependent proliferation response of IL-6 dependent cell line KT-3.

BEST MODE FOR CARRYING OUT THE INVENTION

From peripheral B lymphocytes taken from 20 healthy donors, cDNAs ofeach of immunoglobulin heavy (H) chain and light (L) chain wereamplified by RT-PCR and combined together with a linker DNA to preparesingle chain Fv (scFv) DNAs where the VH chain and VL chain DNAs fromlymphocytes of healthy donors were in random combination.

The scFv DNAs were incorporated into phagemid vector pCANTAB5E toprepare a scFv display phage library consisting of 10⁹ clones fromhealthy donors. This library was then combined with a human IL-6immobilized on a solid phase and an anti-human IL-6 Fv display phageclone was recovered, concentrated and screened. As a result, thescreened scFv clone (IL6gk3-2) produced scFv antibody that binds to ahuman IL-6.

The scFv antibody produced by the clone IL6gk3-2, in spite of being asingle chain, specifically bound to a ligand (IL-6) with an affinityequivalent to the usual complete antibody.

The scFv antibody produced by the clone IL6gk3-2, when added to KT-3cell line that proliferates in a human IL-6 dependent manner, inhibitedIL-6 dependent proliferation response of said cell line in aconcentration dependent manner.

The amino acid sequences of VH and VL chains of the above scFv clonehaving the inhibitory activity as well as the nucleotide sequencescoding therefor are indicated in SEQ ID NOs: 1 and 2 (VH chain) and inSEQ ID NOs: 3 and 4 (VL chain), respectively.

In addition, the amino acid sequences of complementarity determiningregions (CDR1 to CDR3), which are included in the above amino acidsequences, of VH and VL chains are shown below. [VH chain] CDR1: Lys TyrTyr Met Ala (SEQ ID NO: 5) CDR2: Thr Ile Ser Asn Ser Gly Asp Ile (SEQ IDNO: 6) Ile Asp Tyr Ala Asp Ser Val Arg Gly CDR3: Glu Tyr Phe Phe Ser PheAsp Val (SEQ ID NO: 7) [VL chain] CDR1: Arg Ala Ser Gln Asp Ile Arg Asn(SEQ ID NO: 8) Trp Val Ala CDR2: Asp Gly Ser Ser Leu Gln Ser (SEQ ID NO:9) CDR3: Gln Gln Ser Asp Ser Thr Pro Ile (SEQ ID NO: 10) Thr Phe

An antibody fragment having a variable region of either the VH chain orthe VL chain as described above or variable regions of both VH and VLchains has a variable region of a human anti-human IL-6 antibody andstrongly interacts with human IL-6 to thereby exert an inhibitoryactivity against the binding between IL-6 and an IL-6 receptor.

Although the VH chain and/or the VL chain of the human anti-human IL-6antibody as disclosed herein were obtained in the form of scFv by thephage antibody technique, the present invention encompasses a humananti-human IL-6 antibody in the form of a complete molecule wherein thedisclosed VH chain and/or VL chain are bound to a constant region of ahuman immunoglobulin, a human anti-human IL-6 antibody fragment such asFab, Fab′ or F(ab′)₂ wherein the disclosed VH chain and/or VL chain arecombined with a portion of a constant region of a human immunoglobulin,and other human anti-human IL-6 antibody fragment such as a humananti-human IL-6 single chain antibody (scAb) wherein scFv is bound to aconstant region of a human immunoglobulin, as well as gene fragmentsencoding these antibodies and the antibody fragments. The presentinvention further encompasses a modified protein molecule wherein a highmolecular weight modifying agent is bound to these antibody and antibodyfragment protein molecules.

INDUSTRIAL APPLICABILITY

As described above, the human anti-human IL-6 antibody and the fragmentmolecules of said antibody according to the present invention mayinhibit various immune responses induced by binding between IL-6 and anIL-6 receptor and hence may be used as an anti-inflammatory, analgesicagent or as a medicament for the treatment and prevention of autoimmunediseases.

Besides, the human anti-human IL-6 antibody and the fragment moleculesof said antibody according to the present invention, in view of theirproperty, may provide an immunological measurement for detection ormeasurement of IL-6 expressing cells in human peripheral blood or inmuscles.

In addition, the human anti-human IL-6 antibody and the fragmentmolecules of said antibody according to the present invention mayfurther provide many other applications when complexed with animmunoadsorbent consisting of an immunologically inactive adsorbent. Forinstance, IL-6 present in human peripheral blood may be purified withimmunoaffinity chromatography. Such an immunoadsorbent complex may alsobe used for purification of IL-6 in a culture supernatant produced byculture cells transformed by the genetic recombination.

Besides, peptides of the variable region of the human anti-human IL-6antibody of the present invention and derivatives of said peptides mayprovide a new means for isolating a peptide or an anti-idiotype antibodythat recognizes the human anti-human IL-6 antibody of the presentinvention from a library. The obtained peptides and the anti-idiotypeantibodies and derivatives thereof are expected to be efficacious fortreating acute inflammation due to IL-6 neutralization or autoimmunediseases (Vreugdenhil G. et al., Rheumatol. Int. 1990; 10(3): 127-30;Hirano T. et al., Ric. Clin. Lab. 1989 January-March; 19(1): 1-10).

The present invention is explained in more detail by means of thefollowing Examples but should not be construed to be limited thereto.

EXAMPLE 1 Construction of Phage Library from Healthy Donors

Phage library was constructed as reported by J. D. Marks et al., J. Mol.Biol., 222: 581-597, 1991 with some modification.

Lymphocytes were isolated from peripheral blood taken from 20 healthydonors by sedimentary centrifugation with Ficol, washed thoroughly withPBS and then treated with ISOGEN (NIPPON GENE CO., LTD) to prepare atotal RNA. The obtained total RNA was divided into four samples and fromeach of the samples were prepared cDNAs with primers specific toconstant regions of either human IgG, IgM, κ chain or λ chain usingfirst strand cDNA synthesis kit (Pharmacia biotech). Using each of theobtained cDNAs as a template, each of antibody V region genes wereamplified by polymerase chain reaction (PCR) using primers specific toeither of combinations of VH(γ or μ) and JH, Vκ and Jκ, or Vλand Jλ, asdescribed by Marks et al.

Then, VH (γ or μ) and Vκ, and VH (γ or μ) and Vλ, were linked togetherwith a linker DNA by assembly PCR (McCafferty, J. et al.: AntibodyEngineering—A Practical Approach, IRL Press, Oxford, 1996) to preparesingle chain scFv DNAs. The obtained scFv DNAs were added with NotI andSfiI restriction sites using PCR, electrophoresed on agarose gel andthen purified. The purified scFv DNAs were digested with the restrictionenzymes NotI (Takara) and SfiI (Takara) and then cloned into phagemidpCANTAB5E (Pharmacia). The obtained phagemids pCANTAB5E where scFv DNAwas bound were introduced into E. coli TG1 cells by electroporation foreach of VH(γ)-Vκ, VH(γ)-Vλ, VH(μ)-Vκ, and VH(μ)-Vλ. From the number ofthe transformed TG1 cells, it was assessed that VH(γ)-Vκ, VH(γ)-Vλ,VH(μ)-Vκ and VH(μ)-Vλ exhibited diversity of 1.1×10⁸, 2.1×10⁸, 8.4×10⁷and 5.3×10⁷ clones, respectively. With M13KO7 helper phage, phageantibodies were expressed on the transformed TG1 cells to prepare scFvdisplay phage library derived from healthy donors.

EXAMPLE 2 Panning

Human IL-6 was dissolved in 1 mL 0.1M NaHCO₃ and the solution wasincubated in 35 mm dish (Iwaki) at 4° C. overnight to immobilize IL-6.To the dish was added 0.5% gelatin/PBS for blocking at 20° C. for 2hours and then the dish was washed six times with 0.1% Tween20-PBS. Tothe dish was then added 0.9 mL of the single chain antibody displayphage solution (1×10¹² tu/mL of the antibody phage library derived fromhealthy donors) for reaction.

After washing the dish ten times with 0.1% Tween20-PBS, 1.0 mL glycinebuffer (pH 2.2) was added to elute single chain antibody display phagesbound to IL-6. After adjusting pH by adding 1M Tris(hydroxymethyl)-aminomethane-HCl, pH9.1, the eluted phages were infectedto E. coli TG1 cells at logarithmic growth phase. The infected TG1 cellswere centrifuged at 3,000×g for 10 minutes. Supernatant was removed,suspended in 200 μL 2×YT culture medium, plated on SOBAG plate (SOBplate containing 2% glucose, 100 μg/ml ampicillin) and then incubatedovernight in an incubator at 30° C. The resulting colonies weresuspended and recovered in a suitable amount of 2×YT culture medium witha scraper (Coastor).

The obtained TG1 solution (50 μL) was inoculated on 30 mL 2×YT culturemedium and rescued with a helper phage to prepare a phage library afterscreening.

For each of the phage libraries VH(γ)-Vκ, VH(γ)-Vλ, VH(μ)-Vκ andVH(μ)-Vλ derived from healthy donors, four pannings in total wereperformed with the IL-6 immobilized plate. After the fourth panning, anyclone was extracted arbitrarily from the SOBAG plate. The scFvexpression was confirmed, specificity was confirmed by IL-6 ELISA and anucleotide sequence was analyzed.

EXAMPLE 3 IL-6 ELISA for Screening

For screening the isolated clones, ELISA was performed as follows: HumanIL-6 and control proteins were immobilized on an ELISA plate forscreening. Each 40 μL/well of a human recombinant IL-6 (1.25 μg/mL), ahuman serum albumin (HSA; 2.5 μg/mL), a human monocyte chemoattractantprotein 1 (MCP-1; 1.25 μg/mL), a human MIP-1α (macrophage inflammatoryprotein 1-α; 1.25 μg/mL) or a human AB blood type serum (1.25 μg/mL)were placed in an ELISA plate (Nunc) which was kept standing at 4° C.for 16 hours for immobilization. The immobilized plate was added with400 μL/well of a PBS solution containing 0.5% BSA, 0.5% gelatin and 5%skimmed milk and was kept standing at 4° C. for 2 hours for blocking.

To the plate was added 40 μL/well of sample solutions containing scFvdisplay phage for reaction. The sample solutions were discarded and theplate was washed with a washing solution five times. The plate wasreacted with biotin-labeled anti-M13 monoclonal antibody (Pharmaciabiotech) and then with anti-mouse IgG antibody labeled with alkalinephosphatase (AP). After washing with a washing solution five times, theplate was added with 50 μL/well of a developing solution of substrate,i.e. a PBS solution containing 1 g/mL p-nitrophenyl phosphate (Wako) and10% diethanolamine (Wako), light-shielded, and developed at roomtemperature to 37° C. for 5 to 10 minutes. Absorbance at 405 nm wasmeasured using Multiplate Autoreader NJ-2001 (Inter Med). As a result,all the clones assessed were confirmed to be specific to IL-6 (FIG. 1).

EXAMPLE 4 Sequence Analysis of Clones

A DNA nucleotide sequence of the isolated clones was determined for scFvgene VH and VL using Dye terminator cycle sequencing FS Ready Reactionkit (Applied Biosystems) As a result of ELISA and sequence analysis, theisolated clones were classified into four classes. Among these, theclone IL6gk3-2 had nucleotide sequences of VH and VL as shown in SEQ IDNOs: 1 and 3, respectively.

EXAMPLE 5 Expression and Recovery of scFv

A soluble scFv was expressed with E. coli HB2151, recovered from E. coliperiplasm fraction and crudely purified. If further purification wasnecessary, affinity purification was performed with RAPAS PurificationModule (Pharmacia Biotech). Purity of the purified scFv protein wasconfirmed by SDS-polyacrylamide gel electrophoresis and Western blottingwhere Etag epitope at the C-terminus of the scFv protein was targeted.For determination of a protein concentration of the purified scFvprotein product, Protein Assay Kit (BIO-RAD) was used.

EXAMPLE 6 Affinity Measurement of Purified scFv by SPR

Using BIAcore (BIAcore), affinity of the purified scFv was measured bySPR. As a result, IL6gk3-2, the clone with the highest affinity amongthe isolated clones, was assessed to have 13×10⁻⁹ M of a dissociationconstant (FIG. 2).

EXAMPLE 7 Effect on Proliferation Response of IL-6 Dependent Cell Line

The purified scFv was assessed for its inhibitory activity on IL-6dependent proliferation response of cell line KT-3 that proliferates inan IL-6 dependent manner. KT-3 cells prepared at 2×10⁴ cells/200 μl/wellwere cultured for four days in the presence of 1.25 to 20 μg/ml of thepurified scFv from the clone IL6gk3-2 and IL-6 (80 pg/ml) and wereassessed for DNA synthesis through thymidine intake. As a result, it wasrevealed that the scFv from the clone IL6gk3-2 inhibited proliferationresponse of KT-3 cells in a concentration dependent manner (FIG. 3).

1. A human anti-human interleukin-6 (hereinafter, referred to as “IL-6”)antibody that binds to human IL-6 and inhibits the biological activitythereof or a fragment of said antibody.
 2. The human anti-human IL-6antibody or a fragment of said antibody of claim 1 which has adissociation constant of 1.0×10⁻⁸ M or less.
 3. A gene fragment codingfor a VH chain of a human anti-human IL-6 antibody that binds to humanIL-6 and inhibits the biological activity thereof.
 4. The gene fragmentof claim 3 wherein complementarity determining regions (CDR1 to CDR3) ofsaid VH chain have the following amino acid sequences: CDR1: Lys Tyr TyrMet Ala (SEQ ID NO: 5) CDR2: Thr Ile Ser Asn Ser Gly Asp Ile Ile (SEQ IDNO: 6) Asp Tyr Ala Asp Ser Val Arg Gly CDR3: Glu Tyr Phe Phe Ser Phe AspVal. (SEQ ID NO: 7)


5. The gene fragment of claim 3 wherein said VH chain has the amino acidsequence depicted in SEQ ID NO:
 2. 6. The gene fragment of claim 5wherein one or several amino acids are deleted, substituted or added inthe amino acid sequence of said VH chain.
 7. A gene fragment coding fora VL chain of a human anti-human IL-6 antibody that binds to human IL-6and inhibits the biological activity thereof.
 8. The gene fragment ofclaim 7 wherein complementarity determining regions (CDR1 to CDR3) ofsaid VL chain have the following amino acid sequences: CDR1: Arg Ala SerGln Asp Ile Arg Asn (SEQ ID NO: 8) Trp Val Ala CDR2: Asp Gly Ser Ser LeuGln Ser (SEQ ID NO: 9) CDR3: Gln Gln Ser Asp Ser Thr Pro Ile (SEQ ID NO:10) Thr Phe.


9. The gene fragment of claim 7 wherein said VL chain has the amino acidsequence depicted in SEQ ID NO:
 4. 10. The gene fragment of claim 9wherein one or several amino acids are deleted, substituted or added inthe amino acid sequence of said VL chain.
 11. A gene fragment coding fora single chain Fv (hereinafter referred to as “scFv”) of a humananti-human IL-6 antibody that binds to human IL-6 and inhibits thebiological activity thereof, said gene fragment consisting of a genefragment coding for a VH chain of said human anti-human IL-6 antibodybound to a gene fragment coding for a VL chain of said human anti-humanIL-6 antibody.
 12. A gene fragment coding for a human anti-human IL-6antibody that binds to human IL-6 and inhibits the biological activitythereof, said gene fragment consisting of a gene fragment coding for aVH chain of said human anti-human IL-6 antibody bound to a humanantibody CH chain gene and a gene fragment coding for a VL chain of saidhuman anti-human IL-6 antibody bound to a human antibody CL chain gene.13. A gene fragment coding for a human anti-human IL-6 antibody fragmentthat binds to human IL-6 and inhibits the biological activity thereof,said gene fragment consisting of a gene fragment coding for a VH chainof said human anti-human IL-6 antibody bound to a portion of a humanantibody CH chain gene and a gene fragment coding for a VL chain of saidhuman anti-human IL-6 antibody bound to a portion of a human antibody CLchain gene.
 14. The gene fragment of claim 13 wherein said antibodyfragment is selected from Fab, Fab′ or F(ab′)₂.
 15. A gene fragmentcoding for a human anti-human IL-6 antibody fragment that binds to humanIL-6 and inhibits the biological activity thereof, said gene fragmentconsisting of the gene fragment coding for the scFv of claim 11 boundeither to a portion of a human antibody CH chain gene or to a portion ofa human antibody CL chain gene.
 16. A human anti-human IL-6 antibodythat binds to human IL-6 and inhibits the biological activity thereof ora fragment of said antibody, which is expressed by the geneticrecombination technique from an expression vector in which the genefragment of any one of claims 3 to 15 is incorporated.
 17. The humananti-human IL-6 antibody or a fragment of said antibody of claim 16which has a dissociation constant of 1.0×10⁻⁸ M or less.
 18. An agentfor inhibiting the binding between IL-6 and an IL-6 receptor comprisingas an active ingredient the human anti-human IL-6 antibody or a fragmentof said antibody of claim
 1. 19. A medicament for preventing or treatinginflammation or immunopathy caused by the binding between human IL-6 anda human IL-6 receptor, said medicament utilizing the agent forinhibiting the binding of claim
 18. 20. The gene fragment of claim 11wherein complementarity determining regions (CDR1 to CDR3) of said VHchain have the following amino acid sequences: CDR1: Lys Tyr Tyr Met Ala(SEQ ID NO: 5) CDR2: Thr Ile Ser Asn Ser Gly Asp Ile Ile (SEQ ID NO: 6)Asp Tyr Ala Asp Ser Val Arg Gly CDR3: Glu Tyr Phe Phe Ser Phe Asp Val(SEQ ID NO: 7)

and/or complementarity determining regions (CDR1 to CDR3) of said VLchain have the following amino acid sequences: CDR1: Arg Ala Ser Gln AspIle Arg Asn (SEQ ID NO: 8) Trp Val Ala CDR2: Asp Gly Ser Ser Leu Gln Ser(SEQ ID NO: 9) CDR3: Gln Gln Ser Asp Ser Thr Pro Ile (SEQ ID NO: 10) ThrPhe.


21. The gene fragment of claim 11 wherein said VH chain has the aminoacid sequence depicted in SEQ ID NO: 2 and/or said VL chain has theamino acid sequence depicted in SEQ ID NO:
 4. 22. The gene fragment ofclaim 21 wherein one or several amino acids are deleted, substituted oradded in the amino acid sequences of said VH chain and/or said VL chain.ID NO: 10).
 23. The gene fragment of claim 12 wherein complementaritydetermining regions (CDR1 to CDR3) of said VH chain have the followingamino acid sequences: CDR1: Lys Tyr Tyr Met Ala (SEQ ID NO: 5) CDR2: ThrIle Ser Asn Ser Gly Asp Ile Ile (SEQ ID NO: 6) Asp Tyr Ala Asp Ser ValArg Gly CDR3: Glu Tyr Phe Phe Ser Phe Asp Val (SEQ ID NO: 7)

and/or complementarity determining regions (CDR1 to CDR3) of said VLchain have the following amino acid sequences: CDR1: Arg Ala Ser Gln AspIle Arg Asn (SEQ ID NO: 8) Trp Val Ala CDR2: Asp Gly Ser Ser Leu Gln Ser(SEQ ID NO: 9) CDR3: Gln Gln Ser Asp Ser Thr Pro Ile (SEQ ID NO: 10) ThrPhe.


24. The gene fragment of claim 12 wherein said VH chain has the aminoacid sequence depicted in SEQ ID NO: 2 and/or said VL chain has theamino acid sequence depicted in SEQ ID NO:
 4. 25. The gene fragment ofclaim 24 wherein one or several amino acids are deleted, substituted oradded in the amino acid sequences of said VH chain and/or said VL chain.26. The gene fragment of claim 13 wherein complementarity determiningregions (CDR1 to CDR3) of said VH chain have the following amino acidsequences: CDR1: Lys Tyr Tyr Met Ala (SEQ ID NO: 5) CDR2: Thr Ile SerAsn Ser Gly Asp Ile Ile (SEQ ID NO: 6) Asp Tyr Ala Asp Ser Val Arg GlyCDR3: Glu Tyr Phe Phe Ser Phe Asp Val (SEQ ID NO: 7)

and/or complementarity determining regions (CDR1 to CDR3) of said VLchain have the following amino acid sequences: CDR1: Arg Ala Ser Gln AspIle Arg Asn (SEQ ID NO: 8) Trp Val Ala CDR2: Asp Gly Ser Ser Leu Gln Ser(SEQ ID NO: 9) CDR3: Gln Gln Ser Asp Ser Thr Pro Ile (SEQ ID NO: 10) ThrPhe.


27. The gene fragment of claim 13 wherein said VH chain has the aminoacid sequence depicted in SEQ ID NO: 2 and/or said VL chain has theamino acid sequence depicted in SEQ ID NO:
 4. 28. The gene fragment ofclaim 27 wherein one or several amino acids are deleted, substituted oradded in the amino acid sequences of said VH chain and/or said VL chain.29. The gene fragment of claim 19 wherein complementarity determiningregions (CDR1 to CDR3) of said VH chain have the following amino acidsequences: CDR1: Lys Tyr Tyr Met Ala (SEQ ID NO: 5) CDR2: Thr Ile SerAsn Ser Gly Asp Ile Ile (SEQ ID NO: 6) Asp Tyr Ala Asp Ser Val Arg GlyCDR3: Glu Tyr Phe Phe Ser Phe Asp Val (SEQ ID NO: 7)

and/or complementarity determining regions (CDR1 to CDR3) of said VLchain have the following amino acid sequences: CDR1: Arg Ala Ser Gln AspIle Arg Asn (SEQ ID NO: 8) Trp Val Ala CDR2: Asp Gly Ser Ser Leu Gln Ser(SEQ ID NO: 9) CDR3: Gln Gln Ser Asp Ser Thr Pro Ile (SEQ ID NO: 10) ThrPhe


30. The gene fragment of claim 15 wherein said VH chain has the aminoacid sequence depicted in SEQ ID NO: 2 and/or said VL chain has theamino acid sequence depicted in SEQ ID NO:
 4. 31. The gene fragment ofclaim 30 wherein one or several amino acids are deleted, substituted oradded in the amino acid sequences of said VH chain and/or said VL chain.32. An agent for inhibiting the binding between IL-6 and an IL-6receptor comprising as an active ingredient the human anti-human IL-6antibody or a fragment of said antibody of claim
 16. 33. A medicamentfor preventing or treating inflammation or immunopathy caused by thebinding between human IL-6 and a human IL-6 receptor, said medicamentutilizing the agent for inhibiting the binding of claim 32.